Molecular Analysis of the 3' Terminal Region of Onion Yellow Dwarf

Phytopathologia Mediterranea (2014) 53, 3, 438−450 DOI: 10.14601/Phytopathol_Mediterr-14027 RESEARCH PAPERS Molecular analysis of the 3’ terminal region of Onion yellow dwarf virus from onion in southern Italy 1,2 3 3 1 1 ARIANA MANGLLI , HEYAM S. MOHAMMED , ADIL ALI EL HUSSEIN , GIOVANNI E. AGOSTEO , GIULIANA ALBANESE 2 and LAURA TOMASSOLI 1 Dipartimento di Agraria, Università degli Studi Mediterranea di Reggio Calabria, Loc. Feo di Vito, 89122, Reggio Calabria, Italy 2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Plant Pathology Research Centre, Via C. G. Bertero 22, 00156, Roma, Italy 3 Department of Botany, Faculty of Science, University of Khartoum, PO Box 321 11115, Khartoum, Sudan Summary. Onion yellow dwarf virus (OYDV) is an economically important pathogen causing severe disease in garlic, onion and other Allium crops. Eleven isolates of OYDV, all from onion originating from Calabria, southern Italy, were genetically analyzed. An OYDV onion isolate from Sudan was also included in this study. The 3’ terminal region of about 2.5 kb of the twelve isolates were sequenced and the sequences comprising a part of the nuclear inclusion a (NIa-Pro), the complete nuclear inclusion b (NIb) and coat protein (CP) genes and the 3’ untranslated region (3’UTR), were compared to each other and to corresponding sequences of other OYDV isolates from different countries and Allium hosts. The within-population nucleotide identity of the Italian OYDV onion isolates was very high (more than 99.3%), whereas nucleotide identity between them and OYDV onion isolates from Germany was 94%, Argentina 92% and Sudan 87%. Recombination analysis among the complete 3’ terminal sequences showed putative recombination breakpoints in the NIb region of the Argentine isolate, with the minor parent related to the Sudanese isolate. Comparison between OYDV isolates from onion and isolates from garlic produced identities of 77-78% for the complete nucleotide region. When the 3’ terminal nucleotide sequence and the complete NIb protein were analyzed, the phylogenetic analysis generated rooted trees with high bootstrap values (100%), showing a genetic grouping into two well separated clades distinctive for onion and garlic isolates of OYDV. Phylogenetic analysis of CP protein and 3’UTR showed lower bootstrap separation values and no distinct sub-grouping of the OYDV isolates from the two major Allium species. Key words: OYDV, Allium cepa, molecular characterization, NIb and CP proteins, 3’UTR region. Introduction hab et al., 2009; Fayad-André et al., 2011; Katis et al., 2012; Kumar et al., 2012; Mohammed et al., 2013). The Onion yellow dwarf virus (OYDV), is one of the virus is reported to be transmitted in a non-persis- most important viruses affectingAllium species (Van tent manner by more than 50 aphid species (Drake et Dijk, 1993). The virus has spread worldwide and a al., 1933), including Myzus persicae (Sulzer), which is high incidence has been found in many countries, the most efficient vector followed by Aphis craccivo- including Greece, Argentina, The Czech Republic, ra (Koch), and A. gossypii (Glover) (Abd El-Wahab, Egypt, Brazil, India and Sudan (Dovas et al., 2001; 2009; Kumar et al., 2011). OYDV was first identified Conci et al., 2003; Klukáčková et al., 2004; Abd El Wa- in onion (Allium cepa L.) in Iowa, USA (Melhus et al., 1929) and has a limited host range restricted to the Al- lium genus (family Amaryllidaceae). It mainly infects Corresponding author: L. Tomassoli onion and garlic (Allium sativum L.), but also shal- E-mail: [email protected] lot (A. ascalonicum L.), leek (A. fistolosum) and other 438 ISSN (print): 0031-9465 www.fupress.com/pm ISSN (online): 1593-2095 © Firenze University Press Molecular variability of Onion yellow dwarf virus from onion Allium species from Asia. No wild species of Allium was first identified only in southern Italy (Dovas and or other weeds have been reported as natural hosts. Vovlas, 2003; Parrella et al., 2005), without providing The main pathways for OYDV survival are therefore published sequences and phylogenetic analysis of volunteer onion plants from previous crops, onion the onion isolates detected. sets or vernalized bulbs for bulb-to-seed crops (Katis In the present study, we identified OYDV in et al., 2012). southern Italy (Calabria) in the onion cultivar ‘Rossa In onion, OYDV causes yellow striping or yellow- di Tropea’ (protected by the IGP European trade- ing, curling, flattening and crinkling of leaves and mark), and the 3’ terminal sequences of eleven OYDV dwarfing of plants. In seed crops, flower stems re- isolates were determined, including part of the NIa- main turgid and round but show yellowing, distor- Pro gene, the complete nuclear inclusion b (NIb) and tion and curling. In addition, they are shorter than coat protein (CP) genes and 3’ untranslated region normal, and produce smaller flowers with reduced (3’UTR). Nucleotide and amino acid sequences were numbers of seeds that are often of poor quality (Ku- examined and compared with those of three OYDV- mar et al., 2012). OYDV infection causes seed yield O isolates, one from Sudan (included in this study) loss up to 50%, reductions in bulb weight and size up and two from Celli et al. (2013). The sequences were to 40%, and impairs seed storage (Conci et al., 2003; also compared with other OYDV-G isolates reported Elnagar et al., 2011; Kumar et al., 2012). in the GenBank database, to determine the similari- Numerous surveys and epidemiological studies ties and differences between the two viral subgroups regarding the garlic strain of OYDV, named OYDV-G of different host origin. Recombination analysis was (Van Dijk, 1993; Katis et al., 2012) have been carried accomplished among the complete 3’ terminal se- out in many countries where the virus causes severe quences of OYDV-O isolates. damage to garlic and other minor Allium spp. (shal- lot, leek and scallion), frequently found in mixed infections with other viral species of the genera Poty- Materials and methods virus, Carlavirus and Allexivirus (Fajardo et al., 2001; Virus isolates Fayad-André et al., 2011; Bagi et al., 2012; Katis et al., 2012; Mohammed et al., 2013). Studies on OYDV in- This study focused on the onion cultivar ‘Rossa fecting onion crops, hereafter designated as OYDV- di Tropea’ (Red of Tropea), grown in the Tropea area O, have also been reported, concerning occurrence, of Calabria (southern Italy), collecting two different distribution and transmission (Hoa et al., 2003; Ku- biotypes of the cultivar characterized by round (in- mar et al., 2011; 2012; Elnagar et al., 2011; Katis et al. dicated as ‘T’) or long (indicated as ‘L’) red bulbs. 2012; Velasquez-Valle et al., 2012; Sevik and Akcura, In the same location, different fields regarding both 2013). bulb (first year: seed-to-bulb) and seed (second OYDV is a 10,538 nucleotide long, single-strand- year: bulb-to-seed) productions were surveyed for ed positive sense RNA virus (GenBank database, OYDV infections during 2012-2013. Samples were Reference accession No: NC005029) of the genus collected from plants showing severe symptoms of Potyvirus (family Potyviridae). The genome encodes yellow striping and crinkling of leaves and distor- a single large polyprotein yielding up to ten mature tion and dwarfing of flower stems (Figure 1). OYDV proteins between the terminal untranslated regions infection was confirmed by DAS-ELISA tests (Loewe (Gibbs and Oshima, 2010), and an overlapping open Biochemica GmbH), and 11 isolates were selected reading frame (ORF), termed pipo, encoding for an for molecular characterization. An OYDV-O isolate eleventh protein (Chung et al., 2008). Information re- (designated O.70) from Sudan, previously identified garding the variability of populations of OYDV has by H.S. Mohammed at Plant Pathology Research been obtained through molecular techniques, and Centre, Rome (Italy), was also included in this study. genetic studies have provided data mainly on garlic isolates, whereas less information is available on on- Viral RNA and RT-PCR assays ion isolates (Arya et al., 2006; Celli et al., 2013). In Italy, the presence of OYDV has long been as- Total RNA was extracted from fresh host leaf tis- sumed, based on symptomathology recorded in on- sue using Real Total RNA from Tissue and Cell kit ion (Marani and Bertaccini, 1983). However, OYDV (Durviz) according to the manufacturer’s instruc- Vol. 53, No. 3, December, 2014 439 A. Manglli et al. Figure 1. Flower stems from an onion seed crop showing distortion and curling caused by OYDV. tions. Primer sets (Table 1) were designed by mul- min, 57°C for 1 min, 72°C for 1 min, and a final ex- tiple nucleotide sequence alignments of OYDV iso- tension at 72°C for 10 min. A genus-specific primer lates from Allium spp. available in GenBank, choos- pair (CPUP/P9502) amplifying the 3’ CP- 3’UTR re- ing highly conserved nucleotide regions within the gion of potyviruses was also used to complete the species. The annealing positions of the primers were 3’ terminal region of the isolates (Van der Vlugt et arranged in order to obtain contiguous fragments al., 1999) under the following conditions: RT at 46°C with the ends overlapping between each one. For for 60 min, initial denaturation at 94°C for 5 min fol- each primer set, single-step RT-PCR amplification lowed by 35 cycles of 94°C for 2 min, 52°C for 2 min, was performed in a total volume of 25 μL, includ- 72°C for 2 min, and final extension at 72°C for 10 min. ing 2 μL of Total RNA, GoTaq buffer 1× (Promega), 2.5 mM each dNTPS, 4 μM of sense and antisense Cloning and sequencing of PCR products primers, 1.2 U of AMV-RT (Promega), 20 U of RNase Out (Life Technologies) 0.75 U GoTaq Polymerase The PCR fragment of one Italian and the Suda- (Promega).

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